Fig. 2

Ptenb, PTEN and PTEN QMA, but not Ptenb C124S rescued the hyperbranching vasculature phenotype.

(A-J) Zebrafish embryos from a tg(kdrl:eGFP) ptena+/- ptenb-/- incross were microinjected at the one-cell stage with 300 pg synthetic mRNA encoding wild type PTEN-mCherry, PTEN-mCherry QMA, Ptenb-mCherry WT or Ptenb-mCherry C124S. At 3dpf the embryos were analyzed for the hyperbranching vessel phenotype by confocal live- imaging on a Leica TCS-SPE microscope (anterior to the left, 20x objective, 2μm z-stacks). Pictures show the trunk region distal from the urogenital opening of representative, genotyped embryos. Non-injected control embryos (control) were included for reference. (A’-J’) A close-up is added on the right side of each image. (G) Remarkably, we found a second phenotype in the PTEN-mCherry QMA injected embryos, consisting of a significantly increased number of lacking or stalled intersegmental vessels (indicated with white arrows). (K) Quantification of the embryos showing the typical ptena-/-ptenb-/- hyperbranching vessel phenotype at 3 dpf (blue bars). In the non-injected control (NIC), approximately 25% of the embryos showed the characteristic phenotype (Mendelian segregation). The percentage of embryos showing the stalled vessel phenotype at 3dpf is also indicated (red bars). The statistical significance of each of the conditions compared to the non-injected control was determined using two-tailed Fisher’s exact test and is indicated in the bar graph (ns = not significant, * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001). (L) Embryos from a ptena+/-ptenb-/- incross were microinjected at the one-cell stage with wild type PTEN, PTEN QMA, wild type Ptenb or Ptenb C124S. At 4dpf, single embryos were cut in half. The trunk region was used for genotyping and the anterior half was lysed and processed for immunoblotting. Lysates from siblings and ptena-/-ptenb-/- embryos were run side by side on gels and blotted. The membranes were probed with phosphospecific anti-pAkt antibody (directed against pSer473), stripped and probed with Akt-specific antibody, stripped and probed with a Tubulin-specific antibody as a loading control. Representative blots are shown.